Vol. 7, 615-628, May 1996 Cell Growth & Differentiation 615 Selective Changes in Laminin Adhesion and a6 34 Integrin Regulation Are Associated with the Initial Steps in Keratinocyte Maturation1 Tamar Tennenbaum,2 Luowei Li, Adam J. Belanger, Luigi M. De Luca, and Stuart H. Yuspa3 Laboratory of Cellular Carcmnogenesis and Tumor Promotion, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 Abstract In skin, the distribution of integrins is compartmentalized. Whereas the a IJ integrin complex is polarized to the basal portion of proliferating cells in the basal layer juxtaposed to the basement membrane, 3I 1 integrin receptors are localized on the cell surface surrounding basal and suprabasal cells, suggesting fi integrins mediate both cell-matrix and cell-cell interactions. As initiation of maturation in skin is associated with the detachment of cells from the basement membrane, the early loss of a I3 , but not a3 1 integrin expression could be a determining factor in the transition from the proliferating to a differentiating keratinocyte. We have studied the regulation of adhesion potential and integrin expression during differentiation of mouse basal keratinocytes cultured in 0.05 m .i Ca2 medium and induced to differentiate in 0.12 m .i Ca2’ medium. Within 12-24 h after elevation of Ca2 , a selective loss of the a6134integrin complex is associated with the induction of the spinous cell marker keratin I . This early differentiation phenotype coincides with loss of cell attachment mediated by a fi to laminins 1 and 5 but not to fibronectin or collagen IV. Selective loss of attachment to laminin is also detected in spinous cells isolated from newborn epidermis in vivo. The loss of creIJ4 protein expression is a consequence of transcriptional and posttranslational events, including reduction in mRNA transcripts, reduced synthesis of the a protein, and enhanced processing of the a and p4 chains as determined by Western blots and pulse- chase experiments in metabolically labeled keratinocytes. Selective processing of the I3 Received 1 1/14/95; revised 2/12/96; accepted 2/26/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1 734 solely to mdi- cate this fact. 1 This work was supported by a grant from Johnson & Johnson Consumer Products, Inc. 2 Present address: Department of Life Sciences, Bar Ilan University, Aamat Gan, Israel. 3 To whom all correspondence should be addressed, at Laboratory of Cellular Carcmnogenesis and Tumor Promotion, National Cancer Institute, Building 37, Room 3B25, 37 Convent Drive MSC 4255, Bethesda, MD 20892-4255. Phone: 301 -496-2162; Fax: 301-496-8709. intracellular domain is detected before loss of I3, from the cell surface in basal keretinocytes, and this process is accelerated during differentiation. Whereas early keratinocyte maturation is linked to the selective loss of the a6134complex, loss of both fi and fi, integrin mRNA and protein occurs as cells proceed to later stages in the differentiation program as induced by 0.5 mM Ca2 or suspension culture. These conditions are characterized by accelerated expression of transglutaminase; reduced keratin 1 protein; loss of adhesion to fibronectin, laminin I , laminin 5, and collagen IV; and rapid cell death. Contributing to the down-regulation of 13, integrins during terminal differentiation is a selective sensitivity of a3131 but not a6134 to down-regulation by transforming growth factors and 1 2’ factors that are also expressed differentially in normal skin. This study indicates that down-regulation of the a J, but not integrins occurs during the initial steps of keratinocyte differentiation and is associated with detachment from the Iaminin matrix. Such changes could contribute an important signal to initiate the process of terminal keratinocyte differentiation. Introduction The integnins are heterodimenic glycoproteins composed of a and f3 subunits expressed in a variety of cell types and involved in cell-matrix and cell-cell interactions (1). Specific combinations of a and j3 heterodimers determine ligand specificity (2-4). However, the same integnins can bind to more than one ligand, and coexpressed integnins can medi- ate the interaction with the same ligand (3, 5). A specialized tissue distribution is associated with the a6 integnin subunit, where a6 associates preferentially with the f3 subunit in epithelial and neuroepithelial cells, while mesenchyme- derived cells express a6f31 (6, 7). The a6f34 integnn complex is a unique receptor among the integnin family. The a6(34 ligand is thought to be a laminin or laminin isoform (6044, 6043). Nevertheless, cells bind to laminins via a6f34 with varying affinities (8-13). The 34 subunit is unique among the p subunits because of its large cyto- plasmic domain composed of 1 000 amino acids compared with 50 amino acids in cytoplasmic domains of other mem- bers of the 13family (6, 14-1 6). Posttranslational modification by proteolytic digestion of the 34 cytoplasmic domain has been described (1 7). Several studies have identified unique intracellular associations of the a6f34 integnin. Whereas most integnins are thought to localize to focal contacts and interact with the actin cytoskeleton, a6 4 is concentrated at the hemidesmosomes of keratinocytes, where it most likely in- teracts with the keratin intermediate filaments (18-22).